(1) Field of invention
This invention relates to the field of nucleic acid chemistry, more specifically to nucleotide analogs, and still more specifically to “non-standard” nucleotide building blocks that, when incorporated into oligonucleotides (DNA or RNA, collectively xNA), present to a complementary strand in a Watson-Crick pairing geometry a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil. Further, this invention relates to nucleic acid analogs that fold in a way that allows them to bind to target molecules by mechanisms other than Watson-Crick hybridization (aptamers), and nucleic acid analogs that chemically transform the molecules that they bind (aptazymes). These are such molecules are often called “aptamers” are “aptazymes”). Most specifically, this invention claims process that involving the binding and/or catalysis by nucleic acid analogs that contain non-standard building blocks where the oligonucleotide themselves is built from building blocks having either the D- or L-absolute configuration.
(2) Description of Related Art
For two decades, many have sought processes that mimic, in the laboratory, biological evolution to select or evolve DNA or RNA (collectively xNA) molecules that act as ligands, receptors, or catalysts. This process has been called Systematic Evolution of Ligands by Exponential Enrichment (SELEX), “in vitro selection”, in vitro evolution, or “laboratory in vitro evolution (LIVE). These process are collectively referred to here as LIVE. The xNA ligands and receptors that bind to a preselected target are often called aptamers. xNA molecules that catalyze a preselected reaction are often called aptazymes.
The literature describing the history of development of LIVE is summarized in the U.S. patent applications Ser. Nos. 13/493,172 and 14/082,800, which rely on technology disclosed in the U.S. patent application Ser. No. 12/999,138. All of these patent applications (collectively called the “parents”) are incorporated in their entireties by reference, including their drawings, abstract, and disclosures, including examples.
As generally'practiced, LIVE generates aptamers or aptazymes by the following steps:
(a) A library of nucleic acid (xNA) molecules (typically 1014 to 1014 different species) is obtained.
(b) The library is then fractionated to create a fraction that contains molecules better able bind to the preselected target(s), or catalyze the preselected reaction(s), than molecules in the fractions left behind. For example, to generate aptamers, this separation can be done by contacting the library with a solid support carrying the target, washing from the support xNA molecules that do not bind, and recovering from the support xNA molecules that have bound. xNA molecules within the library that bind to the target are said to survive the selection.
(c) The surviving xNAs are then used as templates for the polymerase chain reaction (PCR) process. A low level of mutation may be included in the PCR amplification, creating Darwinian “variation” in an in vitro evolution process.
(d) While aptamers/aptazymes having useful binding/catalytic activity may emerge in the first “round” of selection, they generally do not. When they do not, the cycle is repeated. With each cycle of fractionation/selection and PCR amplification, the resulting fraction of xNA molecules becomes more enriched in those that bind to the preselected target or catalyze the preselected reaction.
(e) The product xNA aptamer(s) and aptazyme(s) might be useful if their sequences are not known. However, the utility'of these products is often enhanced if their sequences are known, as this allows them to be generated separately. To obtain those sequences, standard LIVE procedures generally clone the xNA products in their DNA form (either directly for DNA products, or after conversion to a DNA sequence using reverse transcriptase for RNA products) followed by classical sequencing. Alternatively, next generation sequence can be applied to the mixture of survivors. The elements of this approach are reviewed in U.S. Ser. Nos. 13/493,172 and 14/082,800.
U.S. Ser. Nos. 13/493,172 and 14/082,800 also review the many attempts to improve. LIVE with functionalized natural DNA and RNA. However, simply functionalizing standard xNA nucleotides (as in SOMAmers) does not greatly expand its diversity of folds. Nor does it increase the information density of the biopolymer. Further, functionalizing GACT encounters a new set of problems. For example, an xNA molecule having a fluorescent group attached to each nucleobase are hard to make using xNA polymerases. Further, in ways that are not fully understood, having each nucleobase carry a functional group can cause the DNA to cease to follow “rule based” molecular recognition essential for its genetic roles.
U.S. Ser. No. 13/493,172 also discussed how many of the disadvantages of standard LIVE aptamers and aptazymes might be mitigated by expanding the number of nucleotides in DNA. For example, rearranging hydrogen bond donor and acceptor groups on the nucleobases increases the number of independently replicable nucleosides in DNA and RNA from four to twelve (FIG. 1 and FIG. 2). In this “artificially expanded genetic information system” (AEGIS), as many as 12 different nucleotide “letters” pair via as many as six distinguishable hydrogen bonding patterns to give a system that can be copied and evolve like natural DNA, using processes disclosed in the parents. The products are oligonucleotides with higher information density and more functional group diversity than standard DNA or RNA (collectively xNA).
The potential for using AEGIS to support LIVE has been recognized since the proposal of the first AEGIS. Indeed, processes for doing LIVE with certain AEGIS-containing nucleotides were offered by U.S. Pat. No. 5,965,363. However, efforts to implement the process disclosed in that patent failed to work experimentally. Steps (a) and (b) (above) in the LIVE process were possible. Libraries of xNA molecules containing AEGIS components could be prepared, Step (a), and these libraries could be fractionated (Step (b)). However, as discussed in U.S. Ser. No. 13/493,172, polymerases were not available to perform PCR on DNA molecules containing multiple AEGIS nucleotides.